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J. Biol. Chem., Vol. 282, Issue 4, 2423-2432, January 26, 2007

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Membrane Cholesterol Content Modulates ClC-2 Gating and Sensitivity to Oxidative Stress^*

Alexandre Hinzpeter¹, Janine Fritsch², Florence Borot, Stéphanie Trudel, Diane-Lore Vieu, Franck Brouillard, Maryvonne Baudouin-Legros, Jérôme Clain^¶, Aleksander Edelman, and Mario Ollero

From the INSERM, U806, Université Paris-Descartes, FacultédeMédecine René Descartes, F-75015 Paris, Proteomic Core Facility, Institut Fédératif de Recherche 94, Université Paris-Descartes, FacultédeMédecine René Descartes, F-75015 Paris, and ^¶EA209, Université Paris-Descartes, Faculté de Pharmacie, F-75006 Paris, France

ClC-2 is a broadly expressed member of the voltage-gated ClC chloride channel family. In this study, we aimed to evaluate the role of the membrane lipid environment in ClC-2 function, and in particular the effect of cholesterol and ClC-2 distribution in membrane microdomains. Detergent-resistant and detergent-soluble microdomains (DSM) were isolated from stably transfected HEK293 cells by a discontinuous OptiPrep gradient. ClC-2 was found concentrated in detergent-insoluble membranes in basal conditions and relocalized to DSM upon cholesterol depletion by methyl--cyclodextrin. As assessed by patch clamp recordings, relocalization was accompanied by acceleration of the activation kinetics of the channel. A similar distribution and activation pattern were obtained when cells were treated with the oxidant tert-butyl hydroperoxide and after ATP depletion. In both cases activation was prevented by cholesterol enrichment of cells. We conclude that the cholesterol environment regulates ClC-2 activity, and we provide evidence that the increase in ClC-2 activity in response to acute oxidative or metabolic stress involves relocalization of this channel to DSM.

Received for publication, August 29, 2006 , and in revised form, October 30, 2006.

^* This work was supported in part by European Commission Grant MIRG-CT-2004-004153. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Recipient of a scholarship from Vaincre la Mucoviscidose.

² To whom correspondence should be addressed: Faculté de Médecine Necker, INSERM U806, 156 Rue de Vaugirard, 75015 Paris, France. Tel.: 33-1-40-61-56-21; Fax: 33-1-40-61-55-91; E-mail: fritsch{at}necker.fr.

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